Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from adrenocorticotropic hormone-treated rats: reconstitution of the isocitrate response with succinate and low concentrations of isocitrate.

Cholesterol side-chain cleavage in isolated adrenal mitochondria requires unique energy requirements that may determine not only electron transport to P450 but also cholesterol availability. In mitochondria from ACTH-treated rats, two approximately equal pools of reactive cholesterol are indicated by the partial effectiveness of succinate (SU; Type A), and the metabolism of residual cholesterol by 1 mM isocitrate (IC; Type B). Type A metabolism is associated with relatively few initial cholesterol-P450scc complexes and is rapidly and selectively lost when mitochondria are preincubated without an energy source. We now show that cholesterol metabolism supported by IC resolves into equal high and low affinity components (EC50 = 10 and 250 microM) exhibiting, respectively, Type A and Type B characteristics. SU and 50 microM IC, in combination, provided nearly the same activity characteristics as 1 mM IC, including resistance to preincubation and increased turnover of cholesterol-P450scc complexes. Much higher (three to six times) and more sustained pregnenolone formation was seen, with all reductants, following either enhancement of the reactive cholesterol pool or addition of 20-alpha-hydroxycholesterol, indicating that adrenocorticotropic hormone-mitochondria are limited by substrate availability. ATP generation was most effectively supported by SU, and IC was maximally active at 50 microM, emphasizing differences between respiratory and steroidogenic energy requirements. ATP production and the maintenance of uniform suppression after in vivo cycloheximide treatment indicate the integrity of the mitochondrial interaction with all reductants. Inhibitors of SU oxidation (KCN, malonate) strongly inhibited SU-supported cholesterol metabolism but had little effect on SU synergism with IC. Fumarate (but not alpha-ketoglutarate or oxaloacetate) was equally effective as a synergist, but was totally ineffective as a reductant. SU or fumarate, therefore, act by a nonreductive pathway to boost NADPH production from low concentrations of IC. This decrease in apparent Km for IC may be mediated by stimulation of mitochondrial uptake of the reductant through the specific transporters.